Cultivating a Cleaner Future

Providing Australia with a viable plant-based fuel drives the work of Professor Chris Grof.

As a child, Professor Chris Grof idled hours away with his siblings in the family car while his father disappeared into fields to investigate a rare plant he had spotted while driving.

While Grof did not inherit his father's habit for impromptu roadside stops, the third-generation biologist says a love of plant science has been carried through the generations. In Grof, that passion for plants has developed into a quest to engineer a viable biofuel that could reduce Australians' reliance on fossil fuels.

Fossil fuels account for 98 per cent of Australia's transport energy needs but pioneering countries such as Brazil, which uses ethanol derived from sugarcane for about one quarter of its transport, have proved that a more substantial biofuels industry is feasible.

Australia's main source of ethanol for biofuel is sugarcane, a crop Grof studied extensively as a research scientist with the CSIRO for 13 years. His work into carbohydrate metabolism and genetic manipulation in sugarcane has drawn international recognition, resulting in a prestigious Underwood Fellowship from the British Biotechnology and Biological Sciences Council and invitations to address forums in Australia and overseas.

Since arriving in Newcastle three years ago to head up the Plant Science Group, Grof has taken a lead role in a research project to develop sorghum as a biofuel. Both sorghum and sugarcane share the same photosynthetic properties.

"Sugarcane is a recalcitrant plant and quite difficult to work with genetically, but with sorghum there is an incredible amount of genetic variability that can be used to introduce a whole host of different traits to the plant," he explains.

"Sorghum is already widely planted in Australia, so farmers know how to grow it, and it has limited use as a human food source, so there is no conflict with the supply of edible crops.

"It is quite drought-resistant and able to grow on marginal soils, so that means we may be able to produce a crop that does not compete for arable land that is being used to grow food."

Grof is supported by a strong team at the University. Their area of research, plant biology, achieved an Excellence in Research Australia rating of five, the highest score possible, rating their research well above world standard.

An emphasis of his research is developing second-generation biofuels. Second-generation technologies derive fuel from the whole of the plant, including the woody or fibrous parts known as the lignocellulosic matter, rather than extracting just the juice or oil, which was the traditional method of producing ethanol or other biofuels.

The advantages of second-generation fuels are that they increase the yield of fuel from the plant and can be derived either from crops that are not part of the food cycle or from the waste material of food crops, such as the stems, leaves and husks.

By manipulating the genetic make-up of the plant, Grof aims to elevate both the sugar content and biomass in order to maximise its use as a biofuel feedstock.

He has partnered with researchers from the University of Queensland under a project funded by the Australian Research Council and industry partner Pacific Seeds to produce cultivars of sweet sorghum with increased sugar concentration. He and his team also collaborate with research groups at Texas A&M University and India's ICRISAT (International Crops Research institute for the Semi-Arid Tropics).

To facilitate Grof's work, the University of Newcastle has committed to building a super-sized greenhouse, supported by a $500,000 ARC grant, to accommodate sorghum pants, which can grow to five metres in height.

"This is long-term research but it is very important for Australia," Grof says.

"Environmentally it is a priority because we need to move away from fossil fuels but it also offers the prospect of a sustainable domestic industry in fuel production, which has the potential to deliver major benefits for rural communities and the country as a whole."

Career Summary

Biography

Research expertise

The research area that I have operated within since the beginning of my postgraduate studies is broadly encompassed by C4 photosynthesis and carbon partitioning.

Between 1993 and 2007, a major focus of my research has been the development of a fundamental understanding of carbon metabolism/ photoassimilate partitioning and more specifically sucrose transport processes at the more detailed cellular level in the key food and biofuel crop sugarcane. By the application of a directed EST sequencing approach undertaken on the major sucrose storage organ in sugarcane, the stem, a number of ESTs encoding novel transporters were identified and full length cDNA sequences isolated. Three putative hexose transporters have been immunolocalised to the phloem of maturing internodes of the stem and an increase in expression of these transporters coincided with the logarithmic increase in sucrose concentration. The single sucrose transporter identified to date exhibited a novel localisation pattern being present in the cell layer surrounding the vascular bundles and showed a strong spike in expression at the initiation of the logarithmic phase of sucrose accumulation. The development of a high level of expertise in functional characterisation of sugar transporters has been realised through substantial collaboration travel awards from Federal Department of Industry Science and Technology and the Australian Academy of Science. Visits to work in the laboratories of premier transport researchers Professor Wolf Frommer, (University of Tuebingen, Germany and Carnegie Institute, Stanford University, USA) and Dr John Ward, (University of Minnesota) has resulted in functional characterisation of the sugarcane sucrose transporter in heterologous systems including yeast and Xenopus laevis oocytes. In addition to researching sucrose transport, I also have considerable expertise in improving transformation and transgene expression in recalcitrant plant species, skills essential for implementing genetic manipulations in order to dissect the role of candidate genes.

In 2008 I began developing a new research platform using Sorghum as a model biofuels feedstock. In addition to investigating sucrose transport mechanisms I have extended the scope of photoassimilate partitioning to include cell wall construction/deconstruction and composition. My group is currently undertaking leading edge research in the development of FTIR and FPA-FTIR as tools for detailed investigation of cereal grass cell wall composition and the development of predictive models of digestibility using spectral analysis. The application of ionic liquids as a novel and efficient means of biomass pre-treatment for the production of second generation biofuels is also currently being investigated.

Qualifications

PhD, James Cook University

Keywords

C4 photosynthesis

Fourier Transform Infrared spectroscopy

biofuels

carbon partitioning

plant biotechnology

plant physiology

plant sugar transporters

sorghum

sugarcane

Fields of Research

Code

Description

Percentage

030199

Analytical Chemistry not elsewhere classified

5

060299

Ecology not elsewhere classified

20

060799

Plant Biology not elsewhere classified

75

Professional Experience

UON Appointment

Title

Organisation / Department

Professor

University of NewcastleSchool of Environmental and Life SciencesAustralia

Color intensity of raw sugar is, in part, a result of the activity of the enzyme polyphenol oxidase (PPO) acting on phenolic compounds to produce dark colored polymers when sugarc... [more]

Color intensity of raw sugar is, in part, a result of the activity of the enzyme polyphenol oxidase (PPO) acting on phenolic compounds to produce dark colored polymers when sugarcane (Saccharum spp.) is crushed to release the juice. Paler colored sugar has a potential market premium over darker sugar. In an attempt to alter the level of PPO activity in transgenic plants, sense and antisense constructs containing the native sugarcane PPO gene were introduced into sugarcane by biolistics. In a series of field experiments, it was demonstrated that PPO activity among clones correlated significantly with juice color. In laboratory crystallizations of raw sugar using juice derived from clones with high and low PPO activity, the juice with the higher PPO activity produced darker colored crystals. PPO activity was elevated and juice color was darker in all types of transgenic plants. However, clones derived from a sense construct had higher PPO activity than the other transgenic clones, tissue culture control clones, or cultivars. Furthermore, northern blot analysis demonstrated that PPO sense transgenics had much higher levels of PPO transcripts in the stem than other clones. This is the first targeted manipulation of an endogenous metabolic enzyme-encoding gene in sugarcane that leads to altered enzyme activity. Although low PPO lines with good agronomic performance were not generated, this research demonstrates the principle that juice and sugar color are correlated with PPO activity, consistent with the hypothesis that lowering PPO activity in sugarcane could reduce the color intensity of juice and raw sugar.

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promo... [more]

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the "Goldfinger" isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter function. The "selective-shotgun" approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional "linker-scanning" deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the prospect of identifying interacting promoter regions.

Sugarcane accumulates high concentrations of sucrose in the mature stem and a number of physiological processes on-going in maturing stem tissue both directly and indirectly allow... [more]

Sugarcane accumulates high concentrations of sucrose in the mature stem and a number of physiological processes on-going in maturing stem tissue both directly and indirectly allow this process. To identify transcripts that are associated with stem maturation, we compared patterns of gene expression in maturing and immature stem tissue by expression profiling and bioinformatic analysis of sets of stem ESTs. This study complements a previous study of gene expression associated directly with sugar metabolism in sugarcane. A survey of sequences derived from stem tissue identified an abundance of several classes of sequence that are associated with fibre biosynthesis in the maturing stem. A combination of EST analyses and microarray hybridization revealed that genes encoding homologues of the dirigent protein, a protein that assists in the stereospecificity of lignin assembly, were the most abundant and most strongly differentially expressed transcripts in maturing stem tissue. There was also evidence of coordinated expression of other categories of fibre biosynthesis and putative defence- and stress-related transcripts in the maturing stem. This study has demonstrated the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques to highlight the very significant transcriptional investment the maturing stem of sugarcane has placed in fibre biosynthesis and stress tolerance, in addition to its already well-documented role in sugar accumulation.

The ability of sugarcane to accumulate sucrose provides an experimental system for the study of gene expression determining carbohydrate partitioning and metabolism. A sequence su... [more]

The ability of sugarcane to accumulate sucrose provides an experimental system for the study of gene expression determining carbohydrate partitioning and metabolism. A sequence survey of 7242 ESTs derived from the sucrose-accumulating, maturing stem revealed that transcripts for carbohydrate metabolism gene sequences (CMGs) are relatively rare in this tissue. However, within the CMG group, putative sugar transporter ESTs form one of the most abundant classes observed. A combination of EST analysis and microarray and northern hybridization revealed that one of the putative sugar transporter types, designated PST type 2a, was the most abundant and most strongly differentially expressed CMG in maturing stem tissue. PST type 2a is homologous to members of the major facilitator super-family of transporters, possessing 12 predicted transmembrane domains and a sugar transport conserved domain, interrupted by a large cytoplasmic loop. Its transcript was localized to phloem companion cells and associated parenchyma in maturing stem, suggesting a role in sugar translocation rather than storage. In addition, other categories of CMGs show evidence of coordinated expression, such as enzymes involved in sucrose synthesis and cleavage, and a majority of enzymes involved in glycolysis and the pentose phosphate pathway. This study demonstrates the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques for studies of the developmental regulation of metabolic enzymes and potential transporters in sugarcane.

Extraction and assay methods were developed for the determination of both soluble and cell wall invertase activity in sugarcane (Saccharum sp.) from minimal (0.5 g) tissue. Cell w... [more]

Extraction and assay methods were developed for the determination of both soluble and cell wall invertase activity in sugarcane (Saccharum sp.) from minimal (0.5 g) tissue. Cell wall invertase (CWI) was measured using a pellet mix procedure and the pH optima ranged between pH 3.2 and 3.6. The pH optima for the soluble invertases were 4.5 and 7.3 for soluble acid (SAI) and neutral (NI) invertase, respectively. At low pH, acid hydrolysis of sucrose was observed and its spurious effect on measured enzyme activity was removed by the inclusion of additional controls run in parallel, which lacked crude plant extract. Invertase activity was examined in sugarcane tissues of varying ages. In leaves and stem, the SAI activity was greatly reduced in mature tissue extracts. Similarly, the CWI activity was reduced in older leaves. In contrast, a less marked drop in NI activity was observed in extracts from old leaves and activity from stem extracts remained constant irrespective of tissue age. The role of SAI has been linked to growth and differentiation and these observations suggest that CWI may also be intrinsically involved in these processes. NI appears to have a housekeeping role in maintaining hexose concentrations within the cytosol.

Improvement of the sucrose content of commercial sugarcane by conventional breeding has reached a plateau, primarily due to the narrow gene pool, and the potential to introduce no... [more]

Improvement of the sucrose content of commercial sugarcane by conventional breeding has reached a plateau, primarily due to the narrow gene pool, and the potential to introduce novel genes or manipulate native genes to influence metabolism may have significant application. This review reports on progress in developing new, and optimising existing, transformation processes for sugarcane, and confirms that the requisite molecular tools for modifying sugarcane metabolism are as yet poorly developed when compared with those currently being applied to dicotyledonous model and crop species. Drawing from the considerable base of biochemical research into sucrose metabolism in sugarcane, a number of target steps for metabolic manipulation are reviewed. Specifically, we review current research into the physiological and biochemical elucidation of the key processes of sucrose synthesis, transport and cleavage. Given the focus of this review on molecular manipulation, particular emphasis is placed on the status of research into the isolation of genes encoding the key enzymes and transporters in the sucrose accumulation process.

Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of t... [more]

Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a... [more]

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluorÂ¿ Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 Â± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 Âµg/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 Âµg and 2.11 Âµg sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 Âµg mGFP5-ER per mg extractable protein.

The use of the green fluorescent protein (GFP) and in vivo screening for transgenic cells has enabled the first visual selection of transgenic sugarcane (Saccharum L. hybrid) tran... [more]

The use of the green fluorescent protein (GFP) and in vivo screening for transgenic cells has enabled the first visual selection of transgenic sugarcane (Saccharum L. hybrid) transformed by Agrobacterium-mediated techniques. Selection of GFP-positive embryogenic callus transformed using Agrobacterium tumefaciens strain AGL0 was performed by fluorescence microscopy for 6 weeks after transformation. The use of GFP as a screenable marker aided the rapid segregation of individual transformation events and facilitated the initial visual selection of transgenic sugarcane callus without antibiotics, herbicides or an assay. The binary vector used in transformation was pTO134, which contains a synthetic g/p gene (sgfpS65T) and the bialaphos resistance gene (bar) both controlled by CaMV 35S promoters. The appearance of GFP-expressing cells observed on embryogenic callus was 5.3%. Subsequently, GFP-positive calli grew in the presence of a level of the herbicide bialaphos, that was toxic to untransformed calli. GFP-positive shoots were regenerated and one sugarcane plant expressed GFP in the initial stages. Molecular analysis confirmed the presence of the sgfpS65T coding region in the genome of regenerated plants. Visual screening for Agrobacterium-mediated transformation events using GFP was an efficient technique, which may be applied for the transformation of other plants.

Eight different commercial and breeding varieties of sugarcane (Saccharum spp.) grown in controlled conditions were assayed for leaf sucrose-phosphate synthase (SPS) (EC 2.1.4.14) activity and leaf sucrose content. Leaf SPS activity measured at 25Â°C ranged between 0.06 and 0.14 nmol sucrose formed Âµg protein-1 min-1. The cross-varietal average for leaf SPS activity was 0.10 nmol Âµm protein-1 min-1 (equivalent to 63.4 Âµmol h-1 g FW-1 or 17.6 nkat g FW-1) which is consistent with previously published leaf SPS activities for sugarcane; however, previous studies have assayed leaf SPS activity at either 30 or 37Â°C. The range of leaf sucrose content across varieties (5.5-18.0 mg sucrose g FW-1, average 11.3 mg g FW-1) was consistent with all but one of four previously published reports. Leaf SPS activity and leaf sucrose content were significantly correlated across the eight varieties examined (r2=0.877, d.f. = 7, P<0.001). Whilst previous reports have indicated a co-relationship between leaf SPS activity and leaf sucrose content in single sugarcane varieties both diurnally and with different nutrient regimes, this study shows, for the first time, that this co-relationship also holds true across a range of sugarcane varieties.

Plants of the Australian commercial sugarcane varieties Q117 and Q138 were grown to 6 months age in a controlled environment at temperatures of 14, 18, 22 and 26Â°C. The rate of n... [more]

Plants of the Australian commercial sugarcane varieties Q117 and Q138 were grown to 6 months age in a controlled environment at temperatures of 14, 18, 22 and 26Â°C. The rate of node appearance, which equates to the rate of leaf appearance, was significantly correlated with temperature across the temperature range examined. Analysis of the varietal rates of node deposition as a function of time allowed determination of both base temperature for node (hence leaf) appearance and phyllochron. The base temperatures for node appearance were 7.8Â°C for Q 117 and 7.6Â°C for Q138, significantly lower than previously published base temperatures for leaf appearance in sugarcane. During the developmental stages covered by this study, phyllochron differed between the two varieties with Q117 requiring 108.7 Â°Cd per node, whilst Q138 required 126.6Â°Cd per node. This work reinforces the value of controlled environment research as a way of elucidating basic functions of plant growth and development.

A polymerase chain reaction-based approach was used to isolate cDNA clones encoding the E1a subunit of the mitochondrial pyruvate dehydrogenase from higher plants. Putative full-l... [more]

A polymerase chain reaction-based approach was used to isolate cDNA clones encoding the E1a subunit of the mitochondrial pyruvate dehydrogenase from higher plants. Putative full-length clones were identified on the basis of similarity to E1a- sequences from nonplant sources. Southern blot analysis revealed a small family of genes in potato (Solanum tuberosum L.), whereas in cucumber (Cucumis sativus) there are only one or two genes. Tissue-specific variation in the relative amounts of E1a mRNA was observed in northern blot analysis of different potato tissues, with the highest steady-state transcript levels found in floral tissue. Measurement of pyruvate dehydrogenase activity in cucumber cotyledons showed that there is a transient increase to a maximum at 4 to 5 d postimbibition. Western blot analysis revealed that the amount of E1a protein also peaks at this time. Steady-state transcript levels in germinating cucumber cotyledons also show transient accumulation, peaking 2 d postimbibition. These data are consistent with regulation of E1a at the level of transcription and/or mRNA stability in postgerminative cucumber cotyledons.

The aim of this work was to characterize the respiratory metabolism of the greening cotyledons of cucumber (Cucumis sativus L.) during early seedling growth and to investigate how... [more]

The aim of this work was to characterize the respiratory metabolism of the greening cotyledons of cucumber (Cucumis sativus L.) during early seedling growth and to investigate how this is integrated with changes in mitochondrial biogenesis and function. In light-grown cotyledons, lipid mobilization extended from germination to 6 days postimbibition, reaching a maximum at 3 to 4 days postimbibition. The rate of dark oxygen uptake reached a maximum at 2 days postimbibition in dark-grown and 3 days postimbibition in light-grown cotyledons. Development of photosynthetic capacity occurred from 4 to 7 days postimbibition. In dark-grown cotyledons, lipid mobilization extended beyond 7 days postimbibition, and there was no greening or acquisition of photosynthetic competence. Measurements of mitochondrial function indicated that the respiratory capacity of the tissue changed such that during lipid mobilization there was a much greater capacity for the operation of the nondecarboxylating portion of the citric acid cycle (succinate to oxaloacetate), whereas during the development of photosynthetic function the activity of the remainder of the cycle (oxaloacetate to succinate) was induced. Comparison of the maximum capacities for mitochondrial substrate oxidations in vitro with the rates of in vivo substrate oxidations, predicted from the rate of lipid breakdown, indicated that mitochondria in this tissue operate at or below state 4 rates, suggesting limitation by both availability of ADP and substrate.

Investigating a Role for NAC Transcription Factors as Master Regulators of Transfer Cell Development in Arabidopsis ThalianaBiological Sciences, Faculty of Science and Information TechnologyCo-Supervisor

2013

The Development of Setaria Species as a Model System to Investigate Type II Cell Wall Construction, Deconstruction and Biomass Quality TraitsBiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor

2013

The Molecular Characterisation of Vernalisation in Safflower via the Development of Genomic and Transcriptomic ResourcesBiological Sciences, Faculty of Science and Information TechnologyCo-Supervisor

2012

The Toolkit for the Rapid Improvement of Bioenergy Crops: The Cell Wall FractionBiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor

Benefits of Plants, Compost and Inoculation with Plant-Associated Microbes for the Development of a Mine Spoil AnthroposolBiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor

Past Supervision

Year

Research Title / Program / Supervisor Type

2014

Phloem Loading and Unloading Pathways in Sorghum Bicolor: The Role of Sucrose Transporters in Export and Storage of SucroseBiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor